Abstract

High performance liquid chromatography has already been widely used in numerous study areas as an effective and fast technique for separation and analysis. At present, more attentions have been paid especially to its use in multi-dimensional chromatography and proteome researches. It also provided new technology and approach for studies in the clinical monitoring of diseases and early diagnose. For complicated clinical samples, it is critical to choose proper HPLC methods for clinical monitoring and early diagnosis. This study established a new HPLC method for leukemiaproteomics, by comparing differential proteins in normal human serum, plasma, and cell extracts, with samples collected from leukemia serum, leukemia cells, as well as the K562 chronic myelogeneous leukemia cell line. It is consisted by four parts:1. Review. The application of HPLC in clinical medicine and proteomics study was introduced. The importance of proteome study in leukemia research was also summarized.2. 2D-HPLC-MALDI-TOF MS detection of the proteins from the serum of leukemia sufferer. The condition of three different two-dimensional chromatography modes were optimized and compared, the weak-anion exchange (WAX)-reversed phase chromatography (RPLC) mode was chose as the separation method for the study of serum proteome. Using this method, the serum proteins of the leukemia sufferers and healthy persons were separated and then detected by Matrix assisted laser desorption/ionization-time of flight massspectrometry (MALDI-TOF MS), the distinct proteins were found out by comparing the MALDI-TOF MS data.3. HPLC separation- MALDI-TOF MS detection of the proteins from K562 cell. A method that using two-dimensional liquid chromatography-MALDI-TOF MS to study the proteins extracted from cells was established. The proteins of K562 cell line was separated by three modes of RPLC\ WAX-RPLC and size exclusion chromatography (SEC)-RPLC, the chromatographic fractions were collected and then detected by MALDI-TOF MS. From comparing the date of those separation modes a conclusion can be made: Using SEC-RPLC separation mode to establish the protein finger print of K562 cell line, more than 120 kinds of proteins from 2KD to 130KD can be detected. The detected proteins are the most and the required information is also most comprehensive by using this separation mode. The establishing of this method provides a valid way to clinical study of leukemia.4. RPLC separation- MALDI-TOF MS detection of proteins in leukemia cell. Theleukemia proteins derived from plasma of leukemia sufferers and healthy persons were separated by RPLC, and the chromatographic fractions were analyzed by MALDI-TOF MS. The distinct proteins can be found out by comparing the obtained data. The distinct proteins of different kinds of leukemia, leukemia and other mahgnant blood disease can also be found out. The establishment of this method can help and guide to the clinical diagnoses of leukemia.